A barrier to simplified diagnostic testing is that current clinical chemistry technologies require significant sample preparation and handling for the analysis of complex biological samples. Sample preparation is a major bottleneck in diagnostics. Indicator fluorophores for specific biomarkers capable of functioning directly in an analyte's medium (e.g., blood, urine) without sample handling or separation steps would require fewer manipulations, thereby producing quicker results and reducing potential health hazards due to sample handling. However, surprisingly little progress has been made in developing such fluorophores. This is due at least partially to the relative lack of long wavelength probes.
There are relatively few classes of near infrared (NIR) active dyes that are routinely used, and only one NIR dye is currently approved for clinical use. Advantages of NIR dyes include minimal interfering absorption and fluorescence from biological samples, inexpensive laser diode excitation, and reduced scattering and enhanced tissue penetration depth. However, there are only relatively few classes of such dyes readily available. These include the phthalocyanines, cyanine dyes and squaraine dyes. Each class of dye has inherent strengths and limitations. For example, almost all the established groups of long-wavelength fluorophores have very small Stokes shifts (i.e., emission-excitation wavelength differences), e.g., 10 nm (Miller, Springer Ser. Fluoresc., 2008, 5, 147-162). If used in conjunction with a relatively broad band light source, such as an LED, there may be significant scattered light background signal, producing a poor signal:noise ratio.
Previous research has investigated red-shifting xanthene dyes for biodiagnostics and imaging applications. Long-wavelength, xanthene-based dyes have been used in cellular imaging applications. However, their spectral properties do not fall within the useful NIR “blood window” of 700-800 nm, which facilitates analyte detection in blood. Rhodamines are “red” or long-wavelength xanthene dyes. One notable long wavelength xanthene dye is rhodamine 800, which emits at the interface of the red and NIR, a few nanometers above or below 700 nm depending on the solvent. However, it suffers from limited water solubility and dimer formation and a small Stokes shift of 16 nm (Sauer et al., J. Fluoresc., 1995, 5, 247-261), which complicates analysis in blood. Another innovation includes “JA” dyes, which shift the spectra toward longer wavelength through the addition of double bonds to the nitrogen-containing rings. (Sauer et al.; U.S. Pat. No. 5,750,409). Arden-Jacob and co-workers developed an improved series of fluorophores for biodiagnostics in the red region. However, these dyes exhibit rather small Stokes shifts and do not absorb or emit in the NIR (U.S. Pat. No. 5,750,409).
Annulation is another approach used to produce longer wavelength fluorophores. Type [c] annulated xanthenes include seminaphthofluorescein (SNAFL) and seminaphthorhodafluor (SNARF) compound developed by Haugland (Whitaker et al., Anal. Biochem., 1991, 194, 330-344), which have been used as ratiometric pH sensors, metal ion sensors and imaging probes. (Chang et al., PNAS, 2004, 101, 1129-1134; Nolan et al. J. Am. Chem. Soc., 2007, 129, 5910-5918.)